Publication Date

2025

Document Type

Dissertation

Committee Members

David R. Ladle, Ph.D. (Committee Co-Chair); Anthony C. Fries, Ph.D. (Committee Co-Chair); Michael Leffak, Ph.D. (Committee Member); Jeffrey Peters, Ph.D. (Committee Member); Michael Markey, Ph.D. (Committee Member)

Degree Name

Doctor of Philosophy (PhD)

Abstract

Prior to 2014, Enterovirus D68 infections typically caused symptoms resembling the common cold. From 2014-2018, D68 was associated with an increase in acute flaccid myelitis. However, since 2020, neurological complications have all but disappeared. We selected 1076 respiratory specimens previously determined to be positive for rhinovirus or enterovirus from Department of Defense members and their beneficiaries collected globally from October 2018 through January 2024 and underwent sequencing. Of these specimens, 93.7% were identified as rhinoviruses, while 6.3% were enteroviruses, including 30 enterovirus D68. We utilized the Nextstrain bioinformatic pipeline to reconstruct the phylogenetic relationship of these 30 D68 viruses. Twenty-two fell into the B3 clade which was previously associated with neurological complications. The phylogeny and amino acid substitutions suggest globally recognized antigenic shifts that increase immunogenicity. Monitoring the military population and its beneficiaries for D68 and related enterovirus outbreaks is imperative to identify an increase in D68-associated neurologic complications and to support initiatives to surveil polio-like illnesses. To support this, we developed assays capable of detecting Enterovirus and the D68 serotype. We used a molecular approach which utilizes a CRISPR crRNA that hybridizes to the target and activates the Cas nuclease to cleave non-specific RNA linked to a quencher and fluorescent probe to produce a signal. The D68 specific assay was optimized with a limit of detection of 1.2x103 copies/µl. Respiratory specimens confirmed to have enterovirus D68 via sequencing were subjected to the D68 molecular assay that we developed. Specimens above our determined limit of detection resulted in a positive reaction. Specimens positive for enterovirus species via sequencing were subjected to our enterovirus assay with mixed results. Ultimately, our assays were successful but require additional optimization. CRISPR-based molecular assays may be beneficial to our laboratory in the future, especially for organisms with less genetic variability to develop primers and crRNA. In the meantime, our lab will utilize the whole genome sequencing protocol, coupled with NGS viral bioinformatic pipeline, to classify rhinovirus/enterovirus. Respiratory specimens determined to have enterovirus D68 will be subjected to the Nextstrain phylogenetic analysis to further characterize these strains.

Page Count

167

Department or Program

Biomedical Sciences

Year Degree Awarded

2025

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